StemPro® MSC SFM XenoFree offers a completely xeno–free system when used in conjunction with CTS™ CELLstart™ substrate; thus cells are grown in a physiological environment that allows for clinically relevant results.

Complete StemPro® MSC SFM XenoFree Medium can be stored in the dark at 2–8°C for up to 2 weeks.Note: Before use, warm complete medium required for that day at room temperature until it is no longer cool to the touch. Do not warm the medium at 37°C.

Note: Filtering the AB-Human Serum through a 0.22 µm filter is recommended before use.

Complete StemPro® MSC SFM XenoFree Medium with 2.5% human AB serum can be stored in the dark at 2–8°C for up to 2 weeks.Note: Before use, warm complete medium required for that day at room temperature until it is no longer cool to the touch. Do not warm the medium at 37°C.

CTS™ StemPro® MSC SFM Medium can be stored in the dark at 2–8°C for up to 4 weeks, within the expiration date of all three components.Note: Before use, warm complete medium required for that day at room temperature until it is no longer cool to the touch.

Incubate at 36°C to 38°C in a humidified atmosphere of 4–6% CO2 in air for 60–120 minutes. After incubation, remove flasks from the incubator and temporarily place them in a laminar flow hood until use. Immediately before use, remove all CTS™ CELLstart™ Substrate solution and replace with complete medium. Do not rinse coated plates before use. Note: If CTS™ CELLstart™ Substrate coated plates (with coating solution in the plate) are wrapped tightly with Parafilm® sealing film, they can be stored at 2°C to 8°C for 2 weeks. DO NOT REMOVE COATING SOLUTION PRIOR TO STORAGE.

Recovering Cryopreserved Human MSCs

Note: These instructions can be used for either serum-free and xeno-free cultures or for just serum-free cultures by adding the appropriate complete medium in steps 3, 5, 6, and 8. For serum-free, xeno-free cultures, use StemPro® MSC SFM XenoFree Complete Medium and for serum-free cultures, use CTS™ StemPro® MSC SFM Complete Medium.

Rapidly thaw (<1 minute) a frozen vial of human MSCs in a 37°C water bath until a small amount of ice remains.

Pipet the entire contents of the cryovial into a sterile 50-mL conical tube.

Carefully add 5–10 mL of pre-warmed (37°C) complete medium to the conical tube at a rate of approximately 1 drop every 2 seconds and gently swirl after every addition to ensure homogeneity of the cell suspension.

Centrifuge the tubes at 100–200 × g for 5 minutes at room temperature. Aspirate and discard supernatant, being careful not to disturb the cell pellet.

Resuspend the cell pellet in a minimum volume of pre-warmed (37°C) complete medium for cell counting. Determine total viable cell density with a Countess® Automated Cell Counter (alternative automated or manual procedures may be used). Calculate the volume of cell suspension required to seed cells at a density of ≥ 5 × 103 cells/cm2.

Subculturing MSCs

General Guidelines

For xeno-free, serum-free cultures:

StemPro® MSC SFM XenoFree has been developed for the multi-passage expansion of human bone marrow-derived MSCs and Adipose-derived Stem Cells (ADSCs) at greater than clonal densities (≥ 5 × 103 cells/cm2). Reduced seeding densities may result in suboptimal cell expansion, although optimal growth conditions must be determined for each application.

When subculturing human MSCs in StemPro® MSC SFM XenoFree, input cell confluence should be 60–90%, cell viability should be at least 90%, and the growth rate should be in mid-logarithmic phase prior to subculture. Initiating cultures under suboptimal conditions may affect product performance. Transitioning MSCs or ADSCs from serum-containing medium to StemPro® MSC SFM XenoFree does not require an adaptation protocol.

For optimal performance, re-feed the cultures every 2 days with StemPro® MSC SFM XenoFree complete medium.

CTS™ StemPro® MSC SFM has been developed for the primary isolation and multi-passage expansion of human bone marrow-derived MSC and ADSC at greater than clonal densities, with optimal cell expansion observed at ≥ 5 × 103 cells/cm2.

Reduced seeding densities may result in suboptimal cell expansion. Optimal growth conditions must be determined for each application.

It is recommended that human MSC in CTS™ StemPro® MSC SFM be subcultured when cell confluency reaches 60–80%, cells are in mid-logarithmic phase of growth and cell viability is at least 90%. Initiating cultures under suboptimal conditions may affect product performance. Transitioning MSC or ADSC from serum–containing medium to Complete CTS™ StemPro® MSC SFM does not require an adaptation protocol.

For optimal performance and cell growth, cultures should be re-fed every 2 days with fresh complete CTS™ StemPro® MSC SFM.

Propagating MSCs

Observe the stock culture flask (cells growing in current medium formulation or in either complete medium with an inverted microscope and confirm that the cells are ready to be subpassaged (~60–90% confluent for xeno-free, serum-free medium and ~60–80% confluent for serum-free medium).

Add 10 mL of pre-warmed complete medium to a 50 mL conical tube for each flask being harvested.

Aspirate spent medium from the T-75 flask and discard.

Wash the cell monolayer surface with 5–10 mL of CTS™ DPBS without Ca2+ and Mg2+, aspirate, and discard.

Add 3–5 mL of CTS™ TrypLE™ reagent to the T-75 flask, tilt the flask in all directions to ensure complete coverage of cell monolayer. Incubate the cells in CTS™ TrypLE™ reagent for 2 to 10 minutes at 37°C.Note: Cells coming out of serum-containing medium may require a longer incubation time (5 to 10 minutes), while cells growing under serum-free conditions should detach more readily (2 to 3 minutes).

After incubation, check the flask with an inverted microscope for cell detachment. Firmly tap the flask as necessary to facilitate complete cell detachment.

Upon detachment, add 5 mL of pre-warmed StemPro® MSC SFM XenoFree (for xeno-free, serum-free cultures) or 5 mL pre-warmed CTS™ DPBS with calcium and magnesium (for serum-free cultures) to each flask to completely cover the surface area. Transfer cell suspension to a sterile 50-mL conical tube containing the corresponding complete medium. Firmly tap the flask, re-wash with 5 mL of the appropriate complete medium, and collect.Note: The addition of complete medium to harvested cells is critical for preventing the cells from adhering to the wall of the conical tube during centrifugation.

Centrifuge the tubes at 100–200 × g for 5 minutes at room temperature. Aspirate medium and discard being careful not to disturb cell pellet.

Use an automated or manually controlled rate freezing apparatus following standard procedures (1°C decrease per minute) and place the cryovials at –70°C in a cryogenic freezing container (e.g., Mr. Frosty® (1°C) Freezing Container).

After 24 hours, transfer the frozen cells to liquid nitrogen (vapor phase); storage at –200°C to –125°C is recommended.

Appendix A. Expected results

Maintaining trilineage potential under xeno-free conditions

Using StemPro® MSC SFM XenoFree, human MSCs or ADSCs can be expanded for multiple passages while maintaining their multipotent phenotype (i.e., ability to differentiate into osteogenic, chondrogenic and adipogenic lineages) (Figure 3).